The goal of this project is to understand the selection and regulation of autoreactive B lymphocytes in Type 1A or insulin dependent diabetes mellitus (IDDM). In the long term this information will be used to identify new targets for the diagnosis and treatment of the disorder. Clinical and experimental data indicate that IDDM results from loss of self-tolerance and subsequent autoimmune destruction of insulin producing beta cells. Although autoantibodies to insulin, GAD and other islet antigens are recognized as early indicators of loss of immunological tolerance, most studies focus on the role of T lymphocytes in the disease. Several recent studies indicate that B lymphocytes also play a critical role in IDDM. NOD mice, a highly relevant animal model of IDDM, are protected from the disease process when B lymphocytes are blocked or eliminated. Further, data indicate that B lymphocytes are uniquely able to present some key beta cell antigens, and their antigen presenting function includes the ability to govern T cell differentiation. To better understand the role of B lymphocytes in IDDM, NOD mice were produced that express immunoglobulin transgenes (Tg) from anti-insulin mab125. While B lymphocytes that carry anti-insulin transgenes are functionally silenced in normal B6 mice, the same transgenes demonstrate breaches of tolerance when expressed in NOD. In addition, when NOD mice are engineered to harbor only the heavy chain (HC) Tg from mab125 (VH 125Tg), these animals develop diabetes at a significantly faster rate than non-Tg controls. In contrast, NOD mice expressing an otherwise identical transgene (VH281) that differs in only two amino acids that are required for insulin binding are protected from developing diabetes. When these HC-Tg NOD were examined for expression of insulin binding B cells, B cell receptors (BCR) from VH125-Tg NOD were observed to combine with endogenous light chains to produce a heterogeneous population of insulin-binding B cells, some of which bind insulin with high affinity. Tracking anti-insulin B cells in VH125Tg NOD shows that changes in BCR avidity for insulin occurs in concert with progression to diabetes development. These observations indicate that in the context of IDDM, HC-Tg mice provide a unique means to track the fate and function of anti-insulin B cells that are usually buried in a large polyclonal repertoire. The cellular and molecular mechanisms that connect B cell actions to IDDM will be examined in the following specific aims: I. To characterize the shifts in structure and function of a B cell repertoire for insulin that accompanies progressive beta cell destruction in NOD mice. II. To understand the diversity and function of B lymphocytes that invade pancreatic islets in the course of T1DM. III. To determine the requirement for B lymphocytes in the initiation of T cell responses to insulin and proinsulin in the context of T1DM. IV. To characterize the antigen presenting function of B cells whose receptors recognize a beta cell autoantigen.